The present invention relates to annexin derivatives having chelation sites, radiolabeled annexin derivatives, and imaging methods using radiolabeled annexin derivatives.
The formation of vascular thrombi is a significant complicating factor for atherosclerosis and coronary trauma or disease. Techniques for determining the presence of vascular thrombi include invasive techniques that are often cumbersome and fail to detect thrombi with good sensitivity and specificity. Available non-invasive techniques are often of limited value and fail to image arterial thrombi. Common arterial thrombosis imaging use radionuclide methods that are complex and time consuming, and limited in their practical utility. More importantly, these methods generally fail to detect small thrombi which are of significant clinical importance, particularly in coronary arterial thrombi.
Activated platelets associated with vascular thrombi, express phosphatidylserine, an anionic phospholipid, in an amount significantly greater than quiescent platelets, which express little, if any, phosphatidylserine. Annexins are a class of proteins that are characterized by calcium-mediated binding to anionic phospholipids. Annexin V is a human protein of 319 amino acids with a molecular weight of 36,000 Daltons and binds to phosphatidylserine with a high affinity (Kd=7 nM/L). Accordingly, annexin V offers the potential for selective targeting of platelet thrombi. Furthermore, because there is virtually no circulating annexin V endogenous pool to compete for binding sites on thrombi or to dilute exogenously administered annexin V, annexin V is an attractive candidate for the non-invasive detection of vascular thrombi. Recently, radiolabeled annexins derivatives have been used to image vascular thrombi in vivo. Stratton et al., Circulation, 92:3113-3121, 1995.
In addition to its association with vascular thrombi, cell surface expression of phosphatidylserine also occurs during apoptosis. One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Cells undergoing apoptosis redistribute phosphatidylserine from the inner leaflet of the plasma membrane lipid bilayer to the outer leaflet.
Cell death can occur either through necrosis, which results in uncontrolled release of a variety of intracellular substances, or through apoptosis, which is an orchestrated sequence leaving little cellular residue. Through apoptosis, cellular debris is absorbed by neighboring cells without damage to adjacent tissue or extracellular matrix. Apoptosis (or programmed cell death) plays an important role in a number of physiological events including embryogenesis, regulation of the immune system, and homeostasis. Programmed cell death also plays a role in the pathogenesis of a number of disorders including AIDS and other viral illnesses, cerebral and myocardio ischemia, autoimmune and neurodegenerative diseases, organ and bone marrow transplant rejection, and tumor response to chemotherapy and radiation.
Apoptosis has been determined in histological sections with in situ staining of DNA breaks, formed by the cleavage of chromatin by endonucleases, by terminal deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate-biotin nick-and labeling imaging.
Because annexin has a high affinity for cell membranes expressing phosphatidylserine, annexin V derivatives have been utilized to detect apoptosis in hematopoietic cells, neurons, fibroblasts, endothelial cells, smooth muscle cells, carcinomas, lymphomas, all murine embryonic cell types and plant and insect cells. The utility of a radiolabeled annexin V for in vivo imaging of phosphatidylserine expression associated with apoptosis has been reported. Blankenberg et al., Proc. Natl. Acad. Sci. U.S.A., 95:6349-6354, 1998. In the report, phosphatidylserine expression during programmed cell death was detected and imaged through the use of a radiolabeled annexin conjugate (99mTc HYNIC-annexin V). The use of the same radiolabeled annexin conjugate for in vivo imaging of apoptosis during cardiac allograft rejection has also been reported. Vriens et al., The Journal of Thoracic and Cardiovascular Surgery, 116:844-853, 1998.
Despite the benefits and advantages related to the annexin-based imaging agents noted above, a need for improved annexin-based imaging agents that are effective as imaging vascular thrombi and apoptosis and more readily prepared than the currently used annexin conjugates. The present invention seeks to fulfill these needs and provides further related advantages.
In one aspect, the present invention provides a modified annexin having an N-terminal chelation site. By virtue of the chelation site, the modified annexin readily chelates a radionuclide to provide a radiolabeled annexin. In a preferred embodiment, the modified annexin includes an endogenous chelation site that is formed recombinantly. Methods for forming the modified annexin and radiolabeled annexin are also provided.
In another aspect of the present invention, a method for imaging vascular thrombi is provided. In the method, vascular thrombi are imaged using a modified annexin having an N-terminal chelation site to which is complexed a radionuclide.
In a further aspect, the present invention provides a method for imaging apoptosis. In the method, apoptosis is imaged using a modified annexin having an N-terminal chelation site to which is complexed a radionuclide.